Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds

This study characterizes a new, second gene encoding a plastid located glutamine synthetase (GS2) in M. truncatula. The gene encodes a functional GS isoenzyme with unique kinetic properties, which is exclusively expressed in developing seeds. Based on molecular data and the assumption of a molecular clock, it is estimated that the gene arose from a duplication event that occurred about 10 My ago, after legume speciation and that duplicated sequences are also present in closely related species of the Vicioide subclade. Expression analysis by RT-PCR and western blot indicate that the gene is exclusively expressed in developing seeds and its expression is related to seed filling, suggesting a specific function of the enzyme associated to legume seed metabolism. Interestingly, the gene was found to be subjected to alternative splicing over the first intron, leading to the formation of two transcripts with similar open reading frames but varying 5' UTR lengths, due to retention of the first intron. To our knowledge, this is the first report of alternative splicing on a plant GS gene.This study shows that Medicago truncatula contains an additional GS gene encoding a plastid located isoenzyme, which is functional and exclusively expressed during seed development. Legumes produce protein-rich seeds requiring high amounts of nitrogen, we postulate that this gene duplication represents a functional innovation of plastid located GS related to storage protein accumulation exclusive to legume seed metabolism.Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Nitrogen is assimilated in plants through the action of Glutamine Synthetase (GS, EC 6.3.1.2) forming glutamine, which serves as a building block for all nitrogen containing compounds in the plant. GS is a complex and highly regulated enzyme, which in addition to the primary ammonium assimilation, is involved in the reassimilation of ammonium released by a number of